Aladár Pettkó-Szandtner - Academia.edu (original) (raw)

Papers by Aladár Pettkó-Szandtner

International Journal of Molecular Sciences

Due to their low pathogenicity, immunogenicity, and long-term gene expression, adeno-associated v... more Due to their low pathogenicity, immunogenicity, and long-term gene expression, adeno-associated virus (AAV) vectors emerged as safe and efficient gene delivery tools, over-coming setbacks experienced with other viral gene delivery systems in early gene therapy trials. Among AAVs, AAV9 can translocate through the blood-brain barrier (BBB), making it a promising gene delivery tool for transducing the central nervous system (CNS) via systemic administration. Recent reports on the shortcomings of AAV9-mediated gene delivery into the CNS require reviewing the molecular base of AAV9 cellular biology. A more detailed understanding of AAV9’s cellular entry would eradicate current hurdles and enable more efficient AAV9-based gene therapy approaches. Syndecans, the transmembrane family of heparan-sulfate proteoglycans, facilitate the cellular uptake of various viruses and drug delivery systems. Utilizing human cell lines and syndecan-specific cellular assays, we assessed the involvement of sy...

Nature Plants

Symbiotic nitrogen fixation by Rhizobium bacteria in the cells of legume root nodules alleviates ... more Symbiotic nitrogen fixation by Rhizobium bacteria in the cells of legume root nodules alleviates the need for nitrogen fertilizers. Nitrogen fixation requires the endosymbionts to differentiate into bacteroids which can be reversible or terminal. The latter is controlled by the plant, it is more beneficial and has evolved in multiple clades of the Leguminosae family. The plant effectors of terminal differentiation in inverted repeat-lacking clade legumes (IRLC) are nodule-specific cysteine-rich (NCR) peptides, which are absent in legumes such as soybean where there is no terminal differentiation of rhizobia. It was assumed that NCRs co-evolved with specific transcription factors, but our work demonstrates that expression of NCR genes does not require NCR-specific transcription factors. Introduction of the Medicago truncatula NCR169 gene under its own promoter into soybean roots resulted in its nodule-specific expression, leading to bacteroid changes associated with terminal differen...

FEBS Open Bio

Endoplasmic reticulum (ER) stress‐dependent accumulation of incorrectly folded proteins leads to ... more Endoplasmic reticulum (ER) stress‐dependent accumulation of incorrectly folded proteins leads to activation of the unfolded protein response. The role of the unfolded protein response (UPR) is to avoid cell damage and restore the homeostatic state by autophagy; however, excessive ER stress results in apoptosis. Here we investigated the ER stress‐dependent feedback loops inside one of the UPR branches by focusing on PERK‐induced ATF4 and its two targets, called CHOP and GADD34. Our goal was to qualitatively describe the dynamic behavior of the system by exploring the key regulatory motifs using both molecular and theoretical biological techniques. Using the HEK293T cell line as a model system, we confirmed that the life‐or‐death decision is strictly regulated. We investigated the dynamic characteristics of the crucial elements of the PERK pathway at both the RNA and protein level upon tolerable and excessive levels of ER stress. Of particular note, inhibition of GADD34 or CHOP result...

Symbiotic nitrogen fixation by rhizobium bacteria within the cells of legume root nodules allevia... more Symbiotic nitrogen fixation by rhizobium bacteria within the cells of legume root nodules alleviates the need for nitrogen fertilizers. Nitrogen fixation requires the endosymbionts to differentiate into bacteroids and this can be reversible or terminal. The latter is controlled by the plant, is more beneficial and has evolved in a large clade of the legume family. The plant effectors of terminal differentiation are Nodule-specific Cysteine-Rich NCR peptides, which are absent in legumes such as soybean where there is no terminal differentiation of rhizobia. It was assumed that NCRs coevolved with specific transcription factors, but our work demonstrates that expression of NCR genes does not require NCR-specific transcription factors. Introduction of the Medicago truncatula NCR169 gene under its own promoter into soybean roots resulted in nodule-specific expression of NCR169, leading to bacteroid changes associated with terminal differentiation. We identified two AT-Hook Motif Nuclear...

Biopolymers and Cell, 2019

PloS one, 2017

The small GTPases of the Rho family comprising RhoA, Rac1 and Cdc42 function as molecular switche... more The small GTPases of the Rho family comprising RhoA, Rac1 and Cdc42 function as molecular switches controlling several essential biochemical pathways in eukaryotic cells. Their activity is cycling between an active GTP-bound and an inactive GDP-bound conformation. The exchange of GDP to GTP is catalyzed by guanine nucleotide exchange factors (GEFs). Here we report a novel regulatory mechanism of Rac1 activity, which is controlled by a phosphomimetic (Ser179Glu) mutant of syndecan-4 (SDC4). SDC4 is a ubiquitously expressed transmembrane, heparan sulfate proteoglycan. In this study we show that the Ser179Glu mutant binds strongly Tiam1, a Rac1-GEF reducing Rac1-GTP by 3-fold in MCF-7 breast adenocarcinoma cells. Mutational analysis unravels the PDZ interaction between SDC4 and Tiam1 is indispensable for the suppression of the Rac1 activity. Neither of the SDC4 interactions is effective alone to block the Rac1 activity, on the contrary, lack of either of interactions can increase the a...

The Plant Journal, 2005

The Ms;CDKC;1 kinase is structurally similar to those cyclin-dependent kinases (CDKs) that are no... more The Ms;CDKC;1 kinase is structurally similar to those cyclin-dependent kinases (CDKs) that are not involved directly in cell cycle regulation. The presence of a PITAIRE motif in Ms;CDKC;1 suggests that it interacts with cyclins different from known PSTAIRE/PPTALRE kinase regulatory subunits. Here we demonstrate that a Medicago CYCLINT (CYCT) protein is a specific interactor of Ms;CDKC;1 and the interaction between these two proteins gives rise to an active kinase complex that localizes to the nucleus and phosphorylates the carboxy-terminal YSPTSPS heptapeptide repeat domain (CTD) of the largest subunit of RNA polymerase II in vitro. Mutation of Ser to Ala at position 5 within the heptapeptide repeat abolishes substrate phosphorylation by the Ms;CDKC;1 kinase complex. Furthermore, our data show that addition of the Medicago CDKC;1-CYCT;1 heterodimer completely restored the transcriptional activity of a HeLa nuclear extract depleted of endogeneous CDK9 kinase complexes. Together, these results indicate that the Medicago CDKC;1-CYCT;1 complex is a positive regulator of transcription in plants and has a role similar to the CDK9/cyclin T complex of human positive transcription elongation factor P-TEFb.

Cells

The growth of bacterial populations has been described as a dynamic process of continuous reprodu... more The growth of bacterial populations has been described as a dynamic process of continuous reproduction and cell death. However, this is far from the reality. In a well fed, growing bacterial population, the stationary phase inevitably occurs, and it is not due to accumulated toxins or cell death. A population spends the most time in the stationary phase, where the phenotype of the cells alters from the proliferating ones, and only the colony forming unit (CFU) decreases after a while, not the total cell concentration. A bacterial population can be considered as a virtual tissue as a result of a specific differentiation process, in which the exponential-phase cells develop to stationary-phase cells and eventually reach the unculturable form. The richness of the nutrient had no effect on growth rate or on stationary cell density. The generation time seems not to be a constant value, but it depended on the concentration of the starter cultures. Inoculations with serial dilutions of sta...

The Plant Cell

As the gall-inducing smut fungus Ustilago maydis colonizes maize (Zea mays) plants, it secretes a... more As the gall-inducing smut fungus Ustilago maydis colonizes maize (Zea mays) plants, it secretes a complex effector blend that suppresses host defense responses, including production of reactive oxygen species (ROS) and redirects host metabolism to facilitate colonization. We show that the U. maydis effector ROS burst interfering protein 1 (Rip1), which is involved in pathogen-associated molecular pattern (PAMP)-triggered suppression of host immunity, is functionally conserved in several other monocot-infecting smut fungi. We also have identified a conserved C-terminal motif essential for Rip1-mediated PAMP-triggered suppression of the ROS burst. The maize susceptibility factor lipoxygenase 3 (Zmlox3) bound by Rip1 was relocalized to the nucleus, leading to partial suppression of the ROS burst. Relocalization was independent of its enzymatic activity, revealing a distinct function for ZmLox3. Most importantly, whereas Zmlox3 maize mutant plants showed increased resistance to U. maydi...

Journal of plant research, 2015

Rice (Oryza sativa L.) as a model and crop plant with a sequenced genome offers an outstanding ex... more Rice (Oryza sativa L.) as a model and crop plant with a sequenced genome offers an outstanding experimental system for discovering and functionally analyzing the major cell cycle control elements in a cereal species. In this study, we identified the core cell cycle genes in the rice genome through a hidden Markov model search and multiple alignments supported with the use of short protein sequence probes. In total we present 55 rice putative cell cycle genes with locus identity, chromosomal location, approximate chromosome position and EST accession number. These cell cycle genes include nine cyclin dependent-kinase (CDK) genes, 27 cyclin genes, one CKS gene, two RBR genes, nine E2F/DP/DEL genes, six KRP genes, and one WEE gene. We also provide characteristic protein sequence signatures encoded by CDK and cyclin gene variants. Promoter analysis by the FootPrinter program discovered several motifs in the regulatory region of the core cell cycle genes. As a first step towards function...

Arabidopsis pgEBP1-YFP line expressing EBP1-YFP under the EBP1 promoter were grown in 3 repeats a... more Arabidopsis pgEBP1-YFP line expressing EBP1-YFP under the EBP1 promoter were grown in 3 repeats alongside with the same number of repeats of the control 35CaMVS:GFP (ref) for 7 days at 16/8 light and 150-200 seedlings were frozen in liquid N2 and total proteins were extracted in 1 mL of lysis buffer(150 mM NaCl, 1% Igepal CA-630, 50 mM Tris HCl (pH 8.0), 1 mM DTT, 3 mM PMSF, protease inhibitor cocktail, 3mM pNPP 10 uM MG132). The total protein extracts (4mg/IP) were cleared by centrifugation at 13,000g and 4°C for 20 min to remove remaining membrane and DNA, and the supernatant was incubated with 50 µl magnetic beads coupled to monoclonal mouse anti-GFP antibody (Miltenyi Biotec) for 15 min on ice. Magnetic columns were equilibrated using 250 µl lysis buffer. Cell lysates were added to the column after incubation and washed two times with 1 ml ice-cold lysis buffer, five times with 2 ml of 1xPBS (Dulbecco's Phosphate Buffered Saline), and 2 times 1 ml 25 mM ABC (Ammonium bicarbo...

Journal of Experimental Botany, 2021

Csak a novenyek rendelkeznek sejtciklus-specifikusan, a G2/M fazisban expresszalodo ciklin fuggő ... more Csak a novenyek rendelkeznek sejtciklus-specifikusan, a G2/M fazisban expresszalodo ciklin fuggő kinazokkal (CDKB-k). Klonoztuk es funkcionalisan jellemeztuk a lucerna CDKB2;1 kinaz előtti kb. 360 bp hosszu upstream regiot. Bizonyitottuk, hogy ez a szakasz kepes a riporter genek expressziojat az osztodo merisztematikus sejtekre korlatozni transzgen lucerna novenyekben, es G2/M fazis specifikus expressziot biztositani szinkronizalt, lucerna transzgen novenyekből generalt sejtszuszpenzioban. Immunohisztokemiai festessel igazoltuk, hogy transzgen noveny gyokereben a riporter genek az endogen kinaznak megfelelő mintazatban voltak jelen. Megallapitottuk, hogy az eddig szigoruan sejtciklus-specifikus szabalyozasunak hitt CDKB2;1 kinaz a sebzes hatasara is expresszalodik. Az etilen, amely egyik lehetseges sebzes-valasz mediator, szinten bekapcsolta a promotert, de sem a jazminsav, sem a szalicilsav nem hatott a promoter szabalyzasara, amelyek meg lehetseges szignalatvivői lehetnek a sebzes...

New Phytologist, 2020

-In Arabidopsis thaliana phytochrome B (phyB) is the dominant receptor of photomorphogenic develo... more -In Arabidopsis thaliana phytochrome B (phyB) is the dominant receptor of photomorphogenic development under red light. phyB interacts with a set of downstream regulatory proteins, including PHYTOCHROME INTERACTING FACTOR 3 (PIF3). The interaction of PIF3 and the photo-activated phyB leads to the rapid phosphorylation and degradation of PIF3 and also to the degradation of phyB, which events are required for proper photomorphogenesis. -Here we report that PIF3 is SUMOylated on the Lys13 (K13) residue and we could detect this posttranslational modification in a heterologous experimental system and also in planta. -We also found, that the SUMO acceptor site mutant PIF3(K13R) binds stronger to the target promoters than its SUMOylated, wild-type counterpart. Seedlings expressing PIF3(K13R) show elongated hypocotyl response, elevated photoprotection and higher transcriptional induction of red light responsive genes compared with plantlets expressing wild-type PIF3. -These observations are supported by the lower level of phyB in those plants which possess only PIF3(K13R) indicating that SUMOylation of PIF3 alters photomorphogenesis also via the regulation of phyB amount. Conclusively, whereas SUMOylation is generally connected to different stress responses it also fine tunes light signalling by reducing the biological activity of PIF3 thus promoting photomorphogenesis.

Life Science Alliance, 2021

The DNA of all organisms is constantly damaged by physiological processes and environmental condi... more The DNA of all organisms is constantly damaged by physiological processes and environmental conditions. Upon persistent damage, plant growth and cell proliferation are reduced. Based on previous findings that RBR1, the only Arabidopsis homolog of the mammalian tumor suppressor gene retinoblastoma, plays a key role in the DNA damage response in plants, we unravel here the network of RBR1 interactors under DNA stress conditions. This led to the identification of homologs of every DREAM component in Arabidopsis, including previously not recognized homologs of LIN52. Interestingly, we also discovered NAC044, a mediator of DNA damage response in plants and close homolog of the major DNA damage regulator SOG1, to directly interact with RBR1 and the DREAM component LIN37B. Consistently, not only mutants in NAC044 but also the double mutant of the two LIN37 homologs and mutants for the DREAM component E2FB showed reduced sensitivities to DNA-damaging conditions. Our work indicates the exist...

In plants Heat Shock Factors (HSFs) are encoded by large gene families and are primary regulators... more In plants Heat Shock Factors (HSFs) are encoded by large gene families and are primary regulators of responses not only to high temperatures but also to a number of other abiotic stresses and pathogen threats. Here we provide an overview of the diverse world of the plant HSFs through analysis of their functional versatility, regulation and interactions. HSFs can regulate tolerance to a number of extreme conditions including high or low temperatures, drought, hypoxic conditions, soil salinity, toxic minerals, strong irradiation or pathogen defenses. Variability is reflected in expression control with considerable differences in transcript profiles of individual HSF genes. Moreover, alternative splicing and posttranslational modifications provides further variability. HSFs are involved in complex web of protein-protein interactions which include formation of homomeric and heteromeric HSF trimers, and complexes with a number of other regulatory proteins including transcription regulators, chromatin-associated proteins or heat shock proteins (HSPs). Interactions of the Arabidopsis HSFA4A with proteins which control transcription, cellular homeostasis, responses to different stresses and programmed cell death, illustrate the complexity of the regulatory networks related to a plant HSF. Diversity in plant HSFs facilitates the adaptation to multiple adverse environmental conditions, an important feature in response to climate change.

Megallapitottuk, hogy az MsCPK3 a KRPMt 91. szerin aminosavat foszforilalja. Kiserleteink soran b... more Megallapitottuk, hogy az MsCPK3 a KRPMt 91. szerin aminosavat foszforilalja. Kiserleteink soran bebizonyosodott, hogy az A-tipusu CDK-k ugyanezen az aminosavon foszforilaljak a lucerna CDK inhibitor feherjet. A B-tipusu CDK foszforilacio kulonbozőseget vagy azonossagat technikai problemak nem tettek lehetőve, leven ezen kinazokkal nem tudtunk megfelelően erős kinaz aktivitast detektalni a KRPMt foszforilacioja eseten. Tobbszori kiserletek ellenere sem sikerult ubiqitinalast kimutatni sem a vad tipusu sem a foszforilaciot mimikalo rekombinans feherjek eseteben. Elkeszitettuk a 91. szerin aminosav foszforilaciot mimikalo glutamat valamint aszpartat mutansait. Valamint a nem foszforilalhato alanin mutanst is. Ezen rekombinans feherjek egyike sem volt foszforilalhato sem az MsCPK rekombinans feherjevel, sem az A-tipusu CDK-kal. Kinaz gatlasi kiserleteink soran a foszforilaciot mimikalo szerin aszpartat csere jelentos mertekben (ketszeresere) fokozta az inhibitor feherje gatlo kepesseget. Elkeszitettuk a mutans feherjek novenyi expressziojahoz szukseges vektorokat, melyekkel dohany level korongokat transzformaltunk. A GUS pozitiv kalluszok regeneracioja folyamatban van, azonban sajnalatos modon a legigeretesebb, legerősebb expressziot mutato kalluszok eseteben jelentős degeneraciot tapasztaltunk nem kepesek gyokeret letrehozni. A kutatas idő előtt lezarasra kerul, mert kulfoldre tavoztam es a halasztasi kerelmemet nem tamogatta az OTKA Bizottsag Elnoke. | We found that the MsCPK3 Ca-dependent protein kinase could phosphorylate the KRPMt CDK inhibitor on a serine residue at position 91. Our experiments demonstrated that the A-type CDKs phosphorylate the same amino acid residue in alfalfa CDK inhibitor proteins. We were not able to reveal the kinase activity of the B-type CDK on KRPMt due to the fact that we could not purify sufficiently strong activity for clarifying KRPMt phosphorylation event. We made 91 serine phosphorylation mimicking mutants: 91Ser-91Glu and 91Ser-91Asp, and phosphoinactive 91Ser-91Ala mutants as well. None of those recombinant proteins were phosphorylated by MsCPK3 or A-type CDKs. We could not detect ubiqitination and the subsequent degradation of either the wild-type or the phosphorylation mimicking and phosphoinactive recombinant proteins. In kinase inhibition experiments, the 91Ser-91Asp recombinant protein showed significantly (twice) increased inhibitory effect on A-type CDKs. We have created the plant expression vectors with the wild type and mutant KRPMts, which were used for tobacco leaf discs transformation. The regeneration of whole plants from GUS positive calli is in progress, but unfortunately, the most promising calli -showing the strongest expression of GUS - have significant degeneration: they can not form roots. The time before the research is completed, I left for abroad and the application for postponing the project was not supported by the president of the OTKA committee.

Nucleic Acids Research, 2022

Elongation factor TFIIS (transcription factor IIS) is structurally and biochemically probably the... more Elongation factor TFIIS (transcription factor IIS) is structurally and biochemically probably the best characterized elongation cofactor of RNA polymerase II. However, little is known about TFIIS regulation or its roles during stress responses. Here, we show that, although TFIIS seems unnecessary under optimal conditions in Arabidopsis, its absence renders plants supersensitive to heat; tfIIs mutants die even when exposed to sublethal high temperature. TFIIS activity is required for thermal adaptation throughout the whole life cycle of plants, ensuring both survival and reproductive success. By employing a transcriptome analysis, we unravel that the absence of TFIIS makes transcriptional reprogramming sluggish, and affects expression and alternative splicing pattern of hundreds of heat-regulated transcripts. Transcriptome changes indirectly cause proteotoxic stress and deterioration of cellular pathways, including photosynthesis, which finally leads to lethality. Contrary to expecta...